The present invention provides compositions and methods useful in the diagnosis and prognosis of Carney complex (CNC), as well as methods and compositions for the identification of compounds useful in the treatment and/or prevention of CNC. In addition, the present invention provides compositions and methods useful in the diagnosis and treatment of conditions associated with skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. In addition, the present invention provides methods and compositions for the diagnosis and treatment of various types of cancers associated with abnormal activity of protein kinase A. In particular, the present invention provides genetic and other sequence information, as well as assay systems that will find use in these and related areas.
Carney complex (CNC) is a multiple endocrine neoplasia (MEN) syndrome that affects the adrenal cortex, pituitary gland, thyroid gland, and the gonads. It is also associated with skin and mucosa pigmentation abnormalities, as well as myxoid and other neoplasms of mesenchymal and neural crest origin. The syndrome is characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumors, and psammomatous melanotic schwannomas (Carney and Young, Endocrinologist 2:6-21 [1992]; Carney, Semin. Dermatol., 14:90-98 [1995]; Stratakis et al., Am. J. Med. Genet., 80:183-185 [1998]; Carney et al., Mayo Clin. Proc., 61:165-172 [1985]; and Stratakis, Front. Biosci., 5:D353-366 [2000]). The syndrome also belongs to another group of genetic disorders, referred to as the xe2x80x9clentiginosesxe2x80x9d (lentigenoses), which also includes Peutz-Jeghers, LEOPARD, arterial dissections and lentiginosis, Laugier-Hunziker syndrome, Cowden disease, and Ruvalcaba-Myhre-Smith (Bannayan-Zonana) syndrome, and the centrofacial, benign patterned and segmental lentiginoses, all of which have been associated with a variety of developmental defects. The components of the complex have previously been described by the acronyms NAME (nevi, atrial myxomas and ephelides) or LAMB (lentigines, atrial myxomas, and blue nevi) (See e.g., Atherton et al., Br. J. Dermatol., 103:421-429 [1980]; and Rhodes et al., J. Am. Acad. Dermatol., 10,72-82 [1984]). However, it is presently accepted that most, if not all, of these patients had Carney complex (Stratakis, Pediatr. Pathol. Mol. Med., 19:41-68 [2000]).
Although it is relatively rare, the impact of the syndrome on affected patients is significant. As with other multiple neoplasias and lentigenosis syndromes, Carney complex affects many organs and systems. Typically, patients simultaneously have at least two endocrine tumors (e.g., primary pigmented nodular adrenocortical disease [PPNAD], growth hormone and/or prolactin-producing pituitary adenomas, thyroid nodules or carcinomas, testicular neoplasms [primarily large cell calcifying Sertoli cell tumor (LCCSCT)], and ovarian cysts), as well as psammomatous melanotic schwannoma (PMS), epithelioid blue nevus, breast ductal adenoma, and osteochondromyxoma (i.e., a rare bone tumor). The lesions are multicentric, often bilateral in paired organs, and originate in cells of mesenchymal (myxomas) or neural crest origin (spotty skin pigmentation and endocrine tumors). Thus, it is contemplated that the genetic defects associated with Carney complex are involved in the early development, growth, and proliferation of affected cells.
Early reports suggested that Carney complex was an inherited disease of immunological origin (Young et al., N. Eng. J. Med., 321:1659-1664 [1989]; and Berkhout et al., Clin. Endocrinol., 31:185-191 [1989]). Later reports indicated no association with autoimmune disease or immune dysfunction in affected patients (See e.g., Stratakis et al., J. Clin. Invest. 97:699-705). More recently, CNC has been reported to be inherited as an autosomal dominant trait and the responsible genes have been mapped to 2p16 and 17q22-24 (Stratakis et al., J. Clin. Invest., 97:699-705 [1996]; and Casey et al., Circul., 98:2560-2566 [1998]). Nonetheless, the genetic defects responsible for the complex remained unknown, making diagnosis and treatment of Carney complex problematic.
Currently, to make a diagnosis of Carney complex, a patient must either exhibit two of the manifestations listed in Table 1 below, or exhibit one of these manifestations and meet one of the supplemental criteria (i.e., an affected first-degree relative or an inactivating mutation of the PRKAR1A gene).
Additional findings that are suggestive or possibly associated with Carney complex, but are not diagnostic for the disease include: (1) intense freckling (without darkly pigmented spots or typical distribution); (2) blue nevus, usual type (if multiple); (3) cafxc3xa9-au-lait spots or other xe2x80x9cbirthmarksxe2x80x9d; (4) elevated IGF-1 levels, abnormal oGTT, or paradoxical growth hormone responses to TRH testing, in the absence of clinical acromegaly; (5) cardiomyopathy; (6) pilonidal sinus; (7) history of Cushing syndrome, acromegaly, or sudden death in extended family; (8) multiple skin tags and other skin lesions; lipomas; (9) colonic polyps (usually in association with acromegaly); (10) hyperprolactinemia (usually mild and almost always in association with clinical or subclinical acromegaly); (11) single, benign thyroid nodule in a young patient or multiple thyroid nodules in an older patient (detected by ultrasonography); and (12) family history of carcinoma, in particular of the thyroid, colon, pancreas, and the ovary, or other multiple benign or malignant tumors.
Once the above diagnostic criteria have been applied, and a diagnosis has been confirmed, for post-pubertal pediatric and adult patients, the recommended clinical surveillance of patients with Carney complex involves: echocardiograms (annually or biannually for adolescent patients with a history of excised myxoma), testicular ultrasound (annually), thyroid ultrasound (baseline examination; it may be repeated, as needed), transabdominal ultrasound of the ovaries (baseline examination; it may be repeated, as needed); urinary free cortisol levels (annually); and serum IGF-1 levels (annually). For pre-pubertal pediatric Carney complex patients, the recommended clinical surveillance typically involves echocardiograms (annually; biannually for patients with a history of excised myxoma), testicular ultrasound for boys, and close monitoring of growth rate and pubertal staging (annually). In addition to monitoring of urinary free cortisol levels, further evaluation of patients with primary pigmented nodular adrenocortical disease (all age groups) includes monitoring of diurnal cortisol levels (11:30 PM, 12:00 MN, 7:30 AM, and 8:00 AM sampling), dexamethasone-stimulation test (modified Liddle""s test; See, Stratakis et al., Ann. Intern. Med., 131:585-591 [1999]), and adrenal computed tomography. In addition to monitoring of serum IGF-1 levels, further evaluation of Carney complex patients with gigantism/acromegaly (all age groups) includes pituitary magnetic resonance imaging, 3 hour glucose tolerance test (oGTT), and 90 minute TRH testing. For Carney complex patients of all ages with psammomatous melanotic schwannoma, further evaluation includes magnetic resonance imaging (brain, spine, chest, abdomen, retroperitoneum, pelvis).
In view of the vagaries associated with the currently used methods of diagnosis and prognosis of Carney complex, there remains a need in the art for methods and compositions for the diagnosis, prognosis, and treatment of Carney complex, as well as for methods and compositions useful in the identification of compounds suitable for the treatment of Carney complex and the symptoms associated with the disease.
The present invention provides compositions and methods useful in the diagnosis and prognosis of Carney complex (CNC), as well as methods and compositions for the identification of compounds useful in the treatment and/or prevention of CNC. In addition, the present invention provides compositions and methods useful in the diagnosis and treatment of conditions associated with skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. In addition, the present invention provides methods and compositions for the diagnosis and treatment of various types of cancers associated with abnormal activity of protein kinase A. In particular, the present invention provides genetic and other sequence information, as well as assay systems that will find use in these and related areas.
The present invention provides compositions comprising various mutations in the PRKAR1A gene. In particular, the present invention provides the sequence of wild-type exon 2 (SEQ ID NO:27), as well as the amino acid sequence of wild-type exon 2 (SEQ ID NO:28). The present invention also provides exon 2 mutants with the sequence of SEQ ID NO:29, as well as mutants with the sequences of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.
The present invention further provides the sequence of exon 3 (SEQ ID NO:34), as well as the amino acid sequence of wild-type exon 3 (SEQ ID NO:35). The present invention also provides an exon 3 mutant with the sequence of SEQ ID NO:36.
The present invention further provides the sequence of exon 4A (SEQ ID NO:37), as well as the amino acid sequence of wild-type exon 4A (SEQ ID NO:38). The present invention further provides the sequence of exon 4B (SEQ ID NO:39), as well as the amino acid sequence of wild-type exon 4B (SEQ ID NO:40), and an exon 4B mutant (SEQ ID NO:41).
The present invention further provides the sequence of exon 5 (SEQ ID NO:42), as well as the amino acid sequence of wild-type exon 5 (SEQ ID NO:43), and an exon 5 mutant (SEQ ID NO:44).
The present invention also provides the sequence of exon 6 (SEQ ID NO:45), as well as the amino acid sequence of wild-type exon 6 (SEQ ID NO:46). The present invention further provides exon 6 mutants, with the sequences SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.
The present invention further provides the sequence of exon 7 (SEQ ID NO:51), as well as the amino acid sequence of exon 7 (SEQ ID NO:52), and an exon 7 mutant (SEQ ID NO:53).
The present invention also provides the sequence of wild-type exon 8 (SEQ ID NO:54), as well as the amino acid sequence of wild-type exon 8 (SEQ ID NO:55). The present invention also provides exon 8 mutants with the sequences of SEQ ID NO:56, and SEQ ID NO:57.
The present invention further provides the sequence of wild-type exon 9 (SEQ ID NO:58), as well as the amino acid sequence of exon 9 (SEQ ID NO:59), and an exon 9 mutant (SEQ ID NO:60).
The present invention also provides the sequence of wild-type exon 10 (SEQ ID NO:61), as well as the amino acid sequence of wild-type exon 10 (SEQ ID NO:62).
In some embodiments, the present invention provides an isolated nucleotide sequence of the wild-type protein kinase regulatory subunit 1A gene, wherein the sequence is selected from the group consisting of SEQ ID NO:27, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:58, and SEQ ID NO:61.
In other embodiments, the present invention provides an isolated amino acid sequence of the wild-type protein kinase regulatory subunit 1A, wherein the sequence is selected from the group consisting of SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:59, and SEQ ID NO:62.
In further embodiments, the present invention provides a nucleotide sequence of mutant protein kinase regulatory subunit 1A gene, wherein the mutation is selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:60.
In addition, the present invention provides screening methods for detection of mutations in PKA. In some preferred embodiments, the screening methods include, but are not limited to D-HPLC, sequencing, and cDNA PCR amplification. It is contemplated that these methods will find use either alone or in combination, as well as in combination with other diagnostic and prognostic methods. In particularly preferred embodiments, mutations in the genes encoding R1A are detected using the methods of the present invention. However, it is contemplated that the methods of the present invention will find use in the detection and screening of mutations in the other subunits of PKA.
In alternative embodiments, the present invention provides methods and compositions for the detection and screening of subjects suffering from or suspected of suffering from Carney complex and/or skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. In particularly preferred embodiments, these methods include, but are not limited to D-HPLC, sequencing, and cDNA PCR amplification. It is contemplated that these methods will find use either alone or in combination, as well as in combination with other diagnostic and prognostic methods. In particularly preferred embodiments, mutations in the genes encoding R1A are detected using the methods of the present invention. However, it is contemplated that the methods of the present invention will find use in the detection and screening of mutations in the other subunits of PKA.
In other embodiments, present invention provides methods for the detection of mutations in PKA (e.g., for screening and diagnosis) involving restriction digestion of genomic DNA or cDNA obtained from a subject and detection of alterations in the restriction patterns. These alterations in restriction patterns are then correlated with changes in the sequence of the PKA genes. In further embodiments, the samples used in these methods are obtained from subjects who are suffering from or are suspected of suffering from skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. It is contemplated that these methods will find use either alone or in combination, as well as in combination with other diagnostic and prognostic methods. In particularly preferred embodiments, mutations in the genes encoding R1A are detected using the methods of the present invention. However, it is contemplated that the methods of the present invention will find use in the detection and screening of mutations in the other subunits of PKA.
In still further embodiments, the present invention provides methods involving cold or hot SSCP. In preferred embodiments, these methods utilize the primers described herein (e.g., SEQ ID NOS:1-26, and 63-66). In further embodiments, samples used in these methods involve samples obtained from subjects that are suffering from or are suspected of suffering from skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. It is contemplated that these methods will find use either alone or in combination, as well as in combination with other diagnostic and prognostic methods. In particularly preferred embodiments, mutations in the genes encoding R1A are detected using the methods of the present invention. However, it is contemplated that the methods of the present invention will find use in the detection and screening of mutations in the other subunits of PKA.
The present invention also provides methods and compositions for PCR, in particular quantitative PCR (e.g., Taqman) for determination of the relative expression of PRKAR1A mRNA in tumors and/or cell lines. In further embodiments, the mutant tumors and/or cell lines are obtained from subjects who are suffering from or are suspected of suffering from skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. It is contemplated that these methods will find use either alone or in combination, as well as in combination with other diagnostic and prognostic methods. In particularly preferred embodiments, mutations in the genes encoding R1A are detected and analyzed using the methods of the present invention. However, it is contemplated that the methods of the present invention will find use in the detection and screening of mutations in the other subunits of PKA.
In yet other embodiments, the present invention provides protein truncation detection methods which are utilized following expression (e.g., in vitro expression) of cloned mutant PKA DNA. Such protein detection methods are known to those in the art and are suitable for use to detect the truncation mutants of the present invention. In further embodiments, the mutant PKA DNA is obtained from subjects who are suffering from or are suspected of suffering from skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. It is contemplated that these methods will find use either alone or in combination, as well as in combination with other diagnostic and prognostic methods. In particularly preferred embodiments, mutations in the genes encoding R1A are detected using the methods of the present invention. However, it is contemplated that the methods of the present invention will find use in the detection and screening of mutations in the other subunits of PKA.
The present invention also provides methods for detecting a nucleic acid encoding a mutant protein kinase regulatory subunit 1A gene, comprising providing a biological sample from a patient suspected of containing a nucleic acid sequence encoding mutant protein kinase regulatory subunit 1A gene, and a polynucleotide sequence comprising at least ten nucleotides capable of hybridizing to the nucleic acid sequence; hybridizing the polynucleotide sequence to the nucleic acid sequence encoding a mutant protein kinase regulatory subunit 1A gene to produce a hybridization complex; and detecting the hybridization complex. In other embodiments the polynucleotide sequence comprises fewer than ten nucleotides, with the number of nucleotides as desired by the user of the method and/or as appropriate for the particular embodiment of the method used.
In some embodiments of these methods, the mutant protein kinase regulatory subunit 1A gene comprises a mutant exon having a nucleotide sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:60.
In still further embodiments, the methods, further comprise the step of amplifying nucleic acid encoding a mutant protein kinase regulatory subunit 1A gene before the hybridizing step. In some embodiments, amplifying is accomplished using primers selected from the group consisting of SEQ ID NOS:1-26 and SEQ ID NOS:63-66. In additional embodiments, the detecting step comprises the step of detecting the presence of nucleic acid sequence encoding mutant protein kinase regulatory subunit 1A gene. In some embodiments, the detecting is by Northern blotting. In some preferred embodiments, the patient has Carney complex. In some particularly preferred embodiments, the patient is related to at least one individual having Carney complex. In some additional embodiments, the patient exhibits at least one skin pigmentation defect. In still further embodiments, the patient has a least one lesion selected from the group consisting of adrenal tumors, thyroid tumors, pituitary tumors, myxomas, psammomatous melanotic schwannomas and testicular tumors. In other embodiments, the mutant protein kinase regulatory subunit 1A gene is a truncation mutant. In some preferred embodiments, the method further comprises restriction digestion of nucleic acid in the biological sample. In alternative embodiments, the methods further comprise the step of assaying the biological sample from the patient for protein kinase A activity. In still other alternative embodiments, the methods further comprise the step of assaying the biological sample for PK1 inhibition of protein kinase A activity.
The present invention also provides methods for detecting a nucleic acid encoding a mutant protein kinase R1A, comprising the steps of providing: a biological sample from a cell line suspected of containing a nucleic acid sequence encoding mutant protein kinase regulatory subunit 1A gene, and a polynucleotide sequence comprising at least ten nucleotides capable of hybridizing to nucleic acid sequence; hybridizing the polynucleotide sequence to the nucleic acid sequence encoding a mutant protein kinase regulatory subunit 1A gene to produce a hybridization complex; and detecting the hybridization complex. In other embodiments the polynucleotide sequence comprises fewer than ten nucleotides, with the number of nucleotides as desired by the user of the method and/or as appropriate for the particular embodiment of the method used.
In some preferred embodiments, the mutant protein kinase regulatory subunit 1 A gene comprises a mutant exon having a nucleotide sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:60.
In still other embodiments, the methods further comprise the step of amplifying the nucleic acid encoding a mutant protein kinase regulatory subunit 1A gene before the hybridizing step. In some particularly preferred embodiments, amplifying is accomplished using primers selected from the group consisting of SEQ ID NOS:1-26 and SEQ ID NOS:63-66. In additional embodiments, the detecting step comprises the step of detecting the presence of the nucleic acid sequence encoding the mutant protein kinase regulatory subunit 1A gene. In some preferred embodiments, the detecting is by Northern blotting.
In some embodiments of the methods, the cell line is obtained from a patient with Carney complex. In still other embodiments, the cell line is obtained from a patient who is related to at least one individual having Carney complex. In additional embodiments, the cell line is from a patient who exhibits at least one skin pigmentation defect. In further embodiments, the cell line is from a patient with at least one lesion selected from the group consisting of adrenal tumors, thyroid tumors, pituitary tumors, myxomas, psammomatous melanotic schwannomas and testicular tumors. In some embodiments, the cell line is obtained from at least one or more lesion. In additional embodiments, the mutant protein kinase R1A is a truncation mutant. In still further embodiments, the methods further comprise restriction digestion of the nucleic acid in the biological sample. In some preferred embodiments, the methods further comprise the step of assaying the cell line for protein kinase A activity. In some alternative embodiments, the methods further comprise the step of assaying the cell line for PK1 inhibition of protein kinase A activity.
The present invention also provides antibodies directed against protein kinase regulatory subunit 1A protein. In some preferred embodiments, the antibody is directed against wild-type protein kinase regulatory subunit 1A protein, while in other preferred embodiments, the antibody is directed against mutant protein kinase R1A regulatory subunit 1A gene protein.
The present invention also provides immunohistochemistry methods. In some embodiments, these methods involve the use of antibodies directed against PRKAR1A antibody (e.g., the antibodies used in the Examples below), while other embodiments involve the use of indirect methods (e.g., sandwich type assays). It is contemplated that these methods will find use in the detection of samples in which decreased expression of PRKAR1A is suspected. In further embodiments, samples used in these immunohistochemistry methods are from subjects that are suffering from or are suspected of suffering from skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. It is contemplated that these methods will find use either alone or in combination with other diagnostic and prognostic methods. In particularly preferred embodiments, mutant R1As are detected using the methods of the present invention. However, it is contemplated that the methods of the present invention will find use in the detection and screening of mutations in the other subunits of PKA.
The present invention further provides methods for detecting a protein kinase regulatory subunit 1A, comprising: providing a biological sample suspected of containing protein kinase regulatory subunit 1A, and an antibody directed against protein kinase regulatory subunit 1A; exposing the biological sample to the antibody to form a complex comprising protein kinase regulatory subunit 1A bound to the antibody; and detecting the complex.
In some embodiments, the biological sample is from a patient suspected of having Carney complex. In other embodiments, the biological sample is from a patient who is related to at least one individual who has Carney complex.
In some preferred embodiments, the protein kinase regulatory subunit 1A is a mutant protein kinase regulatory subunit 1A. In still further embodiments, the antibody is selected from the group consisting of antibodies directed against wild-type protein kinase regulatory subunit 1A and antibodies directed against mutant protein kinase regulatory subunit 1A. In some preferred embodiments, the method is selected from the group consisting of Western blotting, immunoassays, and immunohistochemistry.
In still further embodiments, the present invention provides screening methods for PKA activity in tumors or cell lines suspected of having at least one PRKAR1A mutation. In some embodiments, the methods comprise cAMP stimulation followed by PKA assay analysis, as well as PK1 inhibition assays of PKA activity. The present invention also provides methods involving the use of Northern blots for detection of mutant PRKAR1A mRNA. However, it is not intended that the present invention be limited to these particular assays, used alone or in combination. In further embodiments, the tumors or cell lines are obtained from subjects who are suffering from or are suspected of suffering from skin pigmentation defects, including but not limited to freckling, as well as endocrine tumors including, but not limited to adrenal and pituitary tumors. It is contemplated that these methods will find use either alone or in combination, as well as in combination with other diagnostic and prognostic methods. In particularly preferred embodiments, mutations in the genes encoding R1A are detected using the methods of the present invention. It is also contemplated that the methods of the present invention will find use in the detection and screening of mutations in the other subunits of PKA. In addition, it is contemplated that the methods of the present invention will find use in the identification and/or characterization of compounds that are effective in treating Carney complex, pigmentation defects, and/or tumors.
The present invention also provides methods and compositions for the identification and/or characterization of compounds useful for the treatment of Carney complex, pigmentation defects, and/or tumors.